EP3177647B1 - Anticorps anti-herg1 - Google Patents

Anticorps anti-herg1 Download PDF

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Publication number
EP3177647B1
EP3177647B1 EP15757443.5A EP15757443A EP3177647B1 EP 3177647 B1 EP3177647 B1 EP 3177647B1 EP 15757443 A EP15757443 A EP 15757443A EP 3177647 B1 EP3177647 B1 EP 3177647B1
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EP
European Patent Office
Prior art keywords
herg1
seq
scfv
molecule
antibody
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Application number
EP15757443.5A
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German (de)
English (en)
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EP3177647A1 (fr
Inventor
Annarosa Arcangeli
Olivia CROCIANI
Silvia CRESCIOLI
Angelica SETTE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universita degli Studi di Firenze
Azienda Ospedaliero Universitaria Careggi
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Universita degli Studi di Firenze
Azienda Ospedaliero Universitaria Careggi
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of antibodies, in particular it relates to anti-hERG1 molecules.
  • the Single Chain Variable Fragment (scFv) is the most popular and one of the smallest recombinant format with an antigen-binding activity function and with the property to be easily manageable for immunological application (Heng & Othman, 2006).
  • a scFv consists of variable regions of heavy (VH) and light (VL) chains, which are joined together by a flexible peptide linker, without compromising the fidelity of the VH-VL paring and antigen-binding sites.
  • the choice of linker can affect the solubility, expression and correct folding of the scFv.
  • Peptide linkers can vary from 10 to 25 amino acids in length and are typically, composed of hydrophilic amino acids such as glycine (G) and serine (S) (Shen et al., 2008). Hydrophilic sequences prevent intercalation of the peptide within or between the variable domains throughout the protein folding (Argos, 1990).
  • hERG1 is a protein which is aberrantly expressed in tumours and other diseases (Arcangeli & Becchetti, 2010). Antibodies against hERG1 have a high potential application in diagnosis and therapy of a large variety of tumours and other diseases which are characterized by an over expression of hERG1.
  • Aim of the present invention is to provide further antibodies against hERG1 in order to overcome some problems related to the in vivo use of murine complete antibodies (immunogenicity and big size of the molecule preventing an efficient permeability).
  • Subject-matter of the present invention is therefore a molecule comprising a Heavy chain Variable (VH) domain having SEQ ID NO:3 and a Light chain Variable domain having SEQ ID NO:4, said molecule having specificity against hERG1 S5-pore extracellular portion.
  • VH Heavy chain Variable
  • mAb-hERG1 was initially structurally well characterised and then engineered to produce a recombinant single chain variable fragment against hERG1 (scFv-hERG1).
  • the main advantage of scFv over intact whole IgG was its small size; scFv dimensions allow it to penetrate more rapidly.
  • the lack of constant regions decreased retention by Fc receptors found in most tissues and organs, which further reduced the side effects.
  • Molecule according to the invention can be a murine or fully humanized mAb.
  • Molecule according to the invention can be a scFv or any other engineered antibody such as Fab, Fv form of simple chain of scFv, diabodies, triabodies, bispecifics, minibodies, phage antibodies.
  • a molecule according to the invention is useful as diagnostic or therapeutic tool.
  • Pathologies which can be diagnosed or treated using a molecule according to the invention are all those pathologies characterized by an over expression or mis-expression of hERG1 protein. Among said pathologies can be listed tumours, neurological diseases, endocrine diseases and neuro-endocrine diseases.
  • a molecule according to the invention in particular the scFV, can also be used as a pharmaceutical delivery vector: so for example it can be covalently on not bonded to radionuclide, enzyme, drugs or toxin.
  • compositions comprising a molecule according to the invention.
  • a molecule according to the invention can be prepared by employing nucleotide sequences SEQ ID NO:1 and SEQ ID NO:2 encoding respectively VH (SEQ ID NO:3) and VL (SEQ ID NO:4).
  • Particularly preferred according to the invention is a method for preparing a scFv according to the invention, said method comprising the use of nucleotide sequence SEQ ID NO:5 encoding for a scFV having SEQ ID NO:6.
  • the method according to the invention implies recombinant techniques.
  • subject-matter of the present invention are also an expression vector or a plasmid comprising SEQ ID NO:1 and SEQ ID NO:2 as well as genetically modified microorganisms comprising an expression vector according to the invention.
  • Yeast supernatant containing scFv-hERG1-6xHis (SEQ ID N: 17), was collected and concentrated with Amicon Ultra-15 10K (Millipore) and purified by chromatography using columns packed with Ni-NTA Agarose resin (Qiagen). Aliquots obtained from purification, marked as Not Bound (NB), Wash (W) and Elution (E), were analyzed through SDS-Page Comassie Staining and therefore by Western Blot, using an anti-6xHis antibody (GeneTex), to verify for the presence of the corresponding scFv-hERG1 (around 30 kDa). Once demonstrated an efficient production of the antibody, the following step was the evaluation of its ability to bind the antigen.
  • mRNA was retro-transcribed into the corresponding cDNA encoding the VH and VL sequences using Random Primers (225 ng, final concentration/reaction) and SuperScript II Reverse Transcriptase (Invitrogen).
  • VH and VL regions For the amplification of VH and VL regions, a 5' primer that anneals to the VH and VL framework 1 (FR1) (primer forward) and a primer that anneals to the constant region adjacent to VH and VL domains (primer reverse) were chosen.
  • VL a degenerate primer able to recognise the kappa light chain was designed.
  • VH a degenerate primer that anneals to the IgG2b heavy chain was designed.
  • Steps 2,3 and 4 were repeated for 24 times.
  • PCR products relative to VH and VL were run on 1% agarose in TAE buffer (Tris, acetic acid and EDTA), then excised from the gel with a scalpel and purified using QIAquick PCR Purification Kit (QIAGEN), according to the manufacturer's instruction.
  • QIAquick Kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes impurities from DNA samples.
  • DH5 ⁇ cells were used for the transformation step and the selected colonies were checked for the presence of the insert in the right orientation.
  • DNA samples were sequenced by PRIMM s.r.l. and the relative products were analysed using ExPASy translation Tool software and basing on Kabat numbering scheme (www.bioinf.org.uk), in order to find the three scFv Complementary Determining Regions (CDR1, CDR2 and CDR3).
  • phagemid vector pHenIX Hoogenboom et al., 1991
  • specific primers containing restriction enzyme recognition sites were used. Primer's sequences are shown below and restriction sites are reported as underlined. When needed, 2 additional bases (reported in blue) to preserve the sequence frame were included.
  • the yeast host Pichia pastoris was used for the scFv-hERG1 expression.
  • the expression cassette was amplified by PCR using primers containing the Fspl and AvrII restriction sites at 3' and 5' ends, respectively.
  • the expression cassette was then cutted with with Fspl and AvrII and cloned into pPIC9K cutted with Eco53KI and AvrII restriction enzymes (NEB).
  • Pichia Pastoris competent cells was transformed by electroporation using 0.2 cm cuvette, 2000V, 400 ohm, 25 ⁇ F. Yeasts were then seeded on MD plates (Minimal Dextrose Medium: 1.34% YNB, 4x10 -5 % biotin, 2% dextrose) + agar and incubated at 30 °C for 3 days.
  • MD plates Minimal Dextrose Medium: 1.34% YNB, 4x10 -5 % biotin, 2% dextrose
  • a protein having SEQ ID N. 16 was expressed by the yeast.
  • the protein having SEQ ID 16 was cut by the yeast at residue 85 before being secreted in the supernatant thus containing a scFv-hERG1-6xHis having SEQ ID N. 17 (corresponding to residues 86-365 of SEQ ID N. 16).
  • ELISA assay was performed on yeast supernatant purified aliquots obtained from Mut+ and MutS yeast following a standard protocol and using an anti-6xHis antibody (GeneTex) 1:500 in PBS + 3%BSA as primary antibody, followed by an anti-rabbit IgG-HRP conjugate antibody (Sigma) 1:500 in PBS + 3%BSA.
  • hERG1 scFv B
  • PANC-1 hERG1 positive cell line
  • PANC-1 cells were grown on glass coverslips and fixed with 4% methanol-free formaldehyde (Thermo Scientific) in PBS. Coverslips were incubated fifteen minutes in PBS 1% SDS at room temperature for antigen retrieval, treated fifteen minutes with 100 mM glycine at room temperature (to quench residual cross-linking activity of the formaldehyde) and permeabilised four minutes with PBS 0.01% Triton X-100. Unspecific binding sites were blocked thirty minutes at room temperature with PBS 10% FBS.
  • coverslips were incubated one hour at room temperature with anti-hERG1 mAb (1 ⁇ g/ml in PBS 10% FBS) followed by 45 minutes incubation at room temperature with Alexa-488 labelled antimouse antibody (Invitrogen) (1:500 in PBS 10% FBS).
  • coverslips were incubated 2 hours at room temperature with scFv hERG1 (1:2 in PBS 10% FBS) .
  • coverslips were incubated 1 hour with anti-6xHis antibody (GeneTex) 1:500 in PBS 10% FBS, followed by 45 minutes incubation with Alexa-488 labelled anti-rabbit antibody (Invitrogen) (1:500 in PBS 10% FBS).
  • hERG1 scFv (B) has the same ability to bind hERG1 positive cells than the full lenght antibody (A).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (13)

  1. Une molécule anti-ERG1 comprenant un domaine variable à chaîne lourde (VH) ayant SEQ ID NO:3 et un domaine variable à chaîne légère ayant SEQ ID NO:4, ladite molécule ayant une spécificité contre la partie extracellulaire des pores S5 hERG1, dans laquelle ladite molécule anti-ERG1 est un fragment variable à chaîne unique (scFV).
  2. La molécule selon la revendication 1, qui est un anticorps modifié ayant un, deux ou plusieurs sites de liaison d'antigène différents.
  3. Le scFv selon la revendication 1 comprenant SEQ ID NO:6.
  4. La molécule selon l'une quelconque des revendications 1 à 3 à utiliser comme médicament.
  5. La molécule selon l'une quelconque des revendications 1 à 3, à utiliser dans le traitement et/ou le diagnostic de pathologies caractérisées par l'expression excessive de hERG1.
  6. La molécule à utiliser selon la revendication 5, dans laquelle les pathologies sont sélectionnées dans le groupe comprenant des tumeurs, des maladies neurologiques, des maladies endocriniennes et des maladies neuroendocriniennes.
  7. Une composition pharmaceutique comprenant une molécule selon l'une quelconque des revendications 1-3.
  8. Une séquence nucléotidique comprenant SEQ ID NO:1 et SEQ ID NO:2 codant respectivement VH et VL selon la revendication 1.
  9. Séquence nucléotidique selon la revendication 8 et comprenant SEQ ID NO:5.
  10. Un vecteur d'expression comprenant les séquences nucléotidiques selon l'une quelconque des revendications 8-9.
  11. Un micro-organisme génétiquement modifié comprenant un vecteur d'expression selon la revendication 10.
  12. Un procédé de production d'une molécule selon l'une quelconque des revendications 1-3, dans lequel des séquences nucléotidiques selon l'une quelconque des revendications 8-9 sont utilisées.
  13. Un procédé de production du scFv selon la revendication 3, dans lequel la séquence nucléotidique SEQ ID NO:5 selon la revendication 9 est utilisée.
EP15757443.5A 2014-08-08 2015-08-06 Anticorps anti-herg1 Active EP3177647B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITFI20140189 2014-08-08
PCT/EP2015/068178 WO2016020483A1 (fr) 2014-08-08 2015-08-06 Anticorps anti-herg1

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EP3177647A1 EP3177647A1 (fr) 2017-06-14
EP3177647B1 true EP3177647B1 (fr) 2018-10-10

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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201700083637A1 (it) 2017-07-21 2019-01-21 Univ Degli Studi Di Firenze Nuovi anticorpi

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITFI20060008A1 (it) * 2006-01-10 2007-07-11 Univ Firenze Ibridoma capace di produrre un anticorpo monoclonale anti-herg1, anticorpo monoclonale cosi' prodotto, metodo per la determinazione dei livelli di proteina herg1 e kit per tale determinazione
ITRM20090578A1 (it) * 2009-11-10 2011-05-11 Noi Per Voi Onlus Nuove composizioni per il trattamento di leucemie chemioresistenti e/o di leucemie potenzialmente chemioresistenti.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ELENA LASTRAIOLI ET AL: "hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett's esophagus and can be exploited for a novel endoscopic surveillance", ONCOTARGET, vol. 7, no. 37, 13 September 2016 (2016-09-13), United States, XP055458511, ISSN: 1949-2553, DOI: 10.18632/oncotarget.11149 *

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EP3177647A1 (fr) 2017-06-14

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